Hepatitis C virus (HCV) is one of the leading causes of liver cancer and liver transplants in the United States. While current treatments show considerable promise, many patients worldwide will not benefit from these treatments due to cost and required long-term administration and monitoring. Furthermore, cured patients are not protected from new HCV infection. A protective vaccine would greatly augment the efforts to reduce the global health impact caused by HCV. An immunocompetent animal model would aid development of an effective HCV vaccine, however this effort is impeded by HCV's strict species-tropism, as efficient HCV infection is restricted to only humans and chimpanzees. The experiments proposed in this application are aimed at studying the basic mechanisms by which HCV tropism is regulated, and are based on our hypothesis that HCV species-specific tropism is influenced, at least in part, by differences in the capacity for HCV to enter host cells and suppress innate immune responses in disparate species. We recently observed that ferrets are able to support HCV infection in vivo, but at much lower levels than observed in humans and chimpanzees. We seek to understand what controls viral tropism of HCV during both entry and post-entry events and will explore species-specific blocks to HCV infection in ferrets. Our preliminary studies identified that the ferret version of the tight junction protein occludin (OCLN) does not function efficiently as an HCV entry factor. To define the cellular determinants of ferret OCLN's activity as an HCV entry factor, we will map critical residues that influence its function as an entry factor during HCV entry. We will also select for mutations in the HCV genome that will allow more efficient use of ferret OCLN to identify viral determinants for this restriction. Furthermore, HCV suppression of the innate immune response is influenced by species-specific factors, as we have found that HCV is unable to cleave ferret MAVS. We propose experiments to examine host and viral determinants that influence HCV cleavage of ferret MAVS and determine how inadequate suppression of the innate immune system in ferrets by HCV influences viral replication. By identifying species-specific entry, replication and innate immunity restrictions to HCV infection across a wide range of species and defining how these blocks contribute to HCV viral tropism, we can devise methods for efficient HCV infection in a range of species as well as provide insight on how host-pathogen interactions impact the life cycles and host susceptibility for a range of other viruses with similar replication and immune evasion mechanisms.